Exocyclic adducts are unique DNA modifications resulting from bindin at two sites of bases that normally are involved in hydrogen-boning for maintaining the doublehelical structure of DNA. These adducts have been shown to be formed in rodents upon exposure to carcinogens. Using a sensitive 32P-postlabeling method combined with high performance liquid chromatography, we obtained evidence that 1,N2-propanodeoxyguanosine adducts of acrolein (AdG) and crotonaldehyde (CdG) are present in the liver DNA of humans and rodents without carcinogen treatment. (22). In this study, we used this method to show that AdG and CdG (Fig. 1) are present in the liver DNA of rodents and humans and that the in vivo formation of these adducts appears to be stereoselective. MATERIALS AND METHODS DNA Isolation and Hydrolysis and Collection of AdductFractions by HPLC. Male A/J mice (25-30 g) and Fischer rats (200-230 g) were purchased from Charles River Breeding Laboratories and housed in polycarbonate cages (250C ± 2TC, 50 ± 10% relative humidity, and 12-hr light/dark cycle). These animals were fed the modified AIN-76A diet and tap water ad libitum. After 3 weeks of acclimatization, all animals were sacrificed. Livers were quickly removed, minced, and frozen at -800C until DNA isolation. DNA was isolated by a modified Marmur's procedure (23). DNA samples were stored at -800C until analysis.DNA (105-300 pg) was obtained from livers ofeach offour mice and four rats and from each of five humans (two males of ages 37 and 94 years; three females of ages ranging from 50 to 60 years). Human liver DNA was isolated from autopsy samples and was provided by Regina Santella (Columbia University). The DNA was enzymatically hydrolyzed to 3'-monophosphates (22). The enzyme digest was filtered through a 0.2-gm Acrodisc Mini Spike syringe filter (Gelman) and was analyzed by HPLC system 1. Fractions corresponding to AdG 3'-monophosphate and CdG 3'-monophosphate were collected according to the retention times of the synthetic standards (22). To ensure that the assay was free of contamination, blank samples were obtained after injecting 300 p4 of water prior to the collection of fractions of DNA hydrolysates from each species. The fractions from the Abbreviations: enals, a,jf-unsaturated aldehydes; AdG, acroleinderived 1,N2-propanodeoxyguanosine; CdG, crotonaldehydederived 1,N2-propanodeoxyguanosine.*To whom reprint requests should be addressed. 7491The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A number of promutagenic exocyclic DNA adducts have recently been detected in both humans and rodents without carcinogen treatment. These observations raised questions about their origins and potential significance in carcinogenesis. In this commentary, we present our views pertaining to the in vivo sources of these cyclic adducts, specifically the cyclic propano and etheno adducts. The basis for our discussion comes mainly from the information generated through a span of more than a decade from several laboratories, including ours. This commentary summarizes the data from the chemical and biochemical studies that provide support for the hypothesis that lipid peroxidation is involved in the endogenous formation of these exocyclic adducts.

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At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25–26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by 32P‐postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case‐by‐case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance. Environ. Mol. Mutagen. 35:222–233, 2000 © 2000 Wiley‐Liss, Inc.

Alcoholic beverage consumption is associated with an increased risk of upper gastrointestinal cancer. Acetaldehyde (AA), the first metabolite of ethanol, is a suspected human carcinogen, but the molecular mechanisms underlying AA carcinogenicity are unclear. In this work, we tested the hypothesis that polyamines could facilitate the formation of mutagenic α-methyl-γ-hydroxy-1,N2-propano-2′-deoxyguanosine (Cr-PdG) adducts from biologically relevant AA concentrations. We found that Cr-PdG adducts could be formed by reacting deoxyguanosine with μM concentrations of AA in the presence of spermidine, but not with either AA or spermidine alone. The identities of the Cr-PdG adducts were confirmed by both liquid and gas chromatography-mass spectrometry. Using a novel isotope-dilution liquid chromatography-mass spectrometry assay, we found that in the presence of 5 mM spermidine, AA concentrations of 100 μM and above resulted in the formation of Cr-PdG in genomic DNA. These AA levels are within the range that occurs in human saliva after alcoholic beverage consumption. We also showed that spermidine directly reacts with AA to generate crotonaldehyde (CrA), most likely via an enamine aldol condensation mechanism. We propose that AA derived from ethanol metabolism is converted to CrA by polyamines in dividing cells, forming Cr-PdG adducts, which may be responsible for the carcinogenicity of alcoholic beverage consumption.

Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1,N 2 -propanodeoxyguanosine adducts (Acr-dG). Studies show that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the present study, we developed a quantitative 32 P-postlabeling/solid phase extraction/HPLC method by optimizing the solid phase extraction and the 32 P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1 fmole. Using this assay, we found Acr-dG can be formed as an artifact during the assay. We obtained evidence from mass spectrometry showing that Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, we detected Acr-dG as a contaminant in the commercial dG and dT 3′-monophosphate samples. Finally, we applied this method to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship.

Our studies found that BRCA1 levels negatively correlate with DNA adducts induced by Benzo(a)pyrene (BaP). Pulse-chase experiments showed that the increase in BaP-induced DNA adducts in BRCA1 knockdown cells may not be associated with BRCA1’s function in nucleotide excision repair activity; rather, it may be associated with its function in modulating transcriptional regulation. BRCA1 knockdown in MCF-10A cells significantly attenuated the induction of CYP1A1 following BaP treatment indicating that the increase in BaP-induced adducts in BRCA1 knockdown cells is not CYP1A1 dependent. However, our study shows that BRCA1 defective cells may still be able to biotransform BaP by regulating other CYP enzymes, including CYP1B1. Knockdown of BRCA1 also severely affected the expression levels of two types of uridine diphosphate glucorunyltransferase (UGT1A1 and UGT1A9) and NRF2. Both UGTs are known as BaP-specific detoxification enzymes, and NRF2 is a master regulator of antioxidant and detoxification genes. Thus, we concluded that the increased amount of BaP-induced DNA adducts in BRCA1 knockdown cells is strongly associated with its loss of functional detoxification. Chromatin immunoprecipitation assay revealed that BRCA1 is recruited to the promoter/enhancer sequences of UGT1A1, UGT1A9, and NRF2. Regulation of UGT1A1 and UGT1A9 expression showed that the induction of DNA adducts by BaP is directly affected by their expression levels. Finally, overexpression of UGTs, NRF2, or ARNT significantly decreased the amount of BaP-induced adducts in BRCA1-deficient cells. Overall, our results suggest that BRCA1 protects cells by reducing the amount of BaP-induced DNA adducts possibly via transcriptional activation of detoxification gene expression.

A 32P-postlabeling method is described that specifically detects and quantifies the 1,N2-propanodeoxyguanosine adducts derived from acrolein (AdG) and crotonaldehyde (CdG) and 1,N2-ethenodeoxyguanosine (EdG) in DNA. These exocyclic adducts are potential DNA lesions caused by exposure to enals as environmental pollutants and as endogenous compounds. This method was developed with the use of the synthetic adduct standards of these exocyclic adducts. The assay relies on HPLC for adduct enrichment prior to labeling and for quantitation and identification after labeling. The labeling efficiencies of adducts at the 1 fmol level ranged from 74 to 96%, whereas they were only 49-60% at the 100 fmol level. This method can detect as low as 0.2 fmol of adduct and allows the detection and quantitative determination of stereoisomers of AdG and CdG. The method was validated by using a sample of enzyme digests of 180 micrograms calf thymus DNA spiked with 25 or 75 fmol of adducts, which is equivalent to 5 or 15 adducts in 10(8) nucleotides. The recovery rates of these adducts in DNA ranged from 30 to 90% at the 25 fmol level and 21 to 55% at the 75 fmol level. Similar to the labeling efficiency, a greater recovery was observed with a lower amount of adduct in DNA. Overall, this method allows the simultaneous identification and quantification of exocyclic adducts AdG, CdG and EdG in DNA. Therefore, it provides a potential tool for studies of the in vivo formation of exocyclic adducts.